Polynucleotide encoding hCDS1, a human cell-cycle checkpoint kinase

ABSTRACT

The invention provides for a novel human checkpoint kinase gene, hCDS1, translated protein, compositions, methods, and kits.

BACKGROUND OF THE INVENTION

The integrity of the genome is of prime importance to a dividing cell.In response to DNA damage, eukaryotic cells rely upon a complex systemof checkpoint controls to delay cell-cycle progression. The normaleukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2,M) which correlate with distinct cell morphology and biochemicalactivity, and cells withdrawn from the cell-cycle are said to be in G0,or non-cycling state. When cells within the cell-cycle are activelyreplicating, duplication of DNA occurs in the S phase, and activedivision of the cell occurs in M phase. See generally Benjamin Lewin,GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA isorganized in the eukaryotic cell into successively higher levels oforganization that result in the formation of chromosomes. Non-sexchromosomes are normally present in pairs, and during cell division, theDNA of each chromosome replicates resulting in paired chromatids. (Seegenerally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB,Chapter 5, 1997).

Checkpoint delays provide time for repair of damaged DNA prior to itsreplication in S-phase and prior to segregation of chromatids in M-phase(Hartwell and Weinert, 1989, Science 246: 629-634). In many cases theDNA-damage response pathways cause arrest by inhibiting the activity ofthe cyclin-dependent kinases (Elledge, 1997, Science, 274: 1664-1671).In human cells the DNA-damage induced G2 delay is largely dependent oninhibitory phosphorylation of Cdc2 (Blasina et al., 1997, Mol. CellBiol., 8: 1-11; Jin et al., 1996, J. Cell Biol., 134: 963-970), and istherefore likely to result from a change in the activity of the opposingkinases and phosphatases that act on Cdc2. However, evidence that theactivity of these enzymes is substantially altered in response to DNAdamage is lacking (Poon et al., 1997, Cancer Res., 57: 5168-5178).

Three distinct Cdc25 proteins are expressed in human cells. Cdc25A isspecifically required for the G1-S transition (Hoffmann et al., 1994,EMBO J., 13: 4302-4310; Jinno et al., 1994, EMBO J. 13: 1549-1556),whereas Cdc25B and Cdc25C are required for the G2-M transition(Gabrielli et al., 1996, J. Cell Sci., 7: 1081-1093; Galaktionov et al.,1991, Cell, 67: 1181-1194; Millar et al., 1991, Proc. Natl. Acad. Sci.USA, 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol., 138:1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phaseprogression is not known.

Much of our current knowledge about checkpoint control has been obtainedfrom studies using budding (Saccharomyces cerevisiae) and fission(Schizosaccharomyces pombe) yeast. A number of reviews of our currentunderstanding of cell cycle checkpoints in yeast and higher eukaryoteshave recently been published (Hartwell & Kastan, 1994, Science 266:1821-1828; Murray, 1994, Current Biology, 6: 872-876; Elledge, 1996,Science, 274: 1664-1672; Kaufmann & Paules, 1996, FASEB J., 10:238-247). In the fission yeast six gene products, rad1⁺, rad3⁺, rad9⁺,rad17⁺, rad26⁺, and hus1⁺ have been identified as components of both theDNA-damage dependent and DNA-replication dependent checkpoint pathways.In addition cds1+ has been identified as being required for theDNA-replication dependent checkpoint and rad27⁺/chk1⁺ has beenidentified as required for the DNA-damage dependent checkpoint in yeast.

Several of these genes have structural homologues in the budding yeastand further conservation across eukaryotes has recently been suggestedwith the cloning of two human homologues of S. pombe rad3⁺: ATM (ataxiatelangiectasia mutated) (Savitsky et al., 1995, Science, 268: 1749-1753)and ATR (ataxia telangiectasia and rad3⁺ related)(Bentley et al, 1996,EMBO J., 15: 6641-6651; Cimprich et al., 1996, Proc. Natl. Acad. Sci.USA, 93: 2850-2855) and of a human homologue of S. pombe rad9⁺(Lieberman et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 13890-13885).

While much is known about yeast checkpoint proteins and genes, thisknowledge is not fully predictive of the existence of correspondinghuman genes or proteins, or their effector role in human cell-cyclecontrol and regulation.

In order to develop new and more effective treatments and therapeuticsfor the amelioration of the effects of cancer, it is important toidentify and characterize human checkpoint proteins and to identifymediators of their activity.

SUMMARY OF THE INVENTION

The present invention is directed to the discovery of a novel humancheckpoint kinase gene hCDS1, protein and constructs and methods for theproduction and use of hCDS1.

In particular, the present invention encompasses a nucleic acid sequencewhich encodes for hCDS1, consisting of the nucleic acid sequence of SEQID NO.: 1. In particular, the invention encompasses the nucleic acidsequence from position 66 to 1694 of the nucleic acid sequence of SEQ IDNO.: 1, which translates into the hCDS1 protein. The present inventionalso encompasses nucleic acid constructs, vectors, plasmids, cosmids andthe like which contain the nucleic acid sequence of SEQ ID NO.: 1. Inparticular, the present invention provides for nucleic acid vectorconstructs which contain the nucleic acid sequence of SEQ ID NO.: 1 andare capable of expressing protein from this nucleic acid sequence. Thepresent invention encompasses nucleic acid vectors that are suitable forthe transformation of host cells, whether eukaryotic or prokaryotic,suitable for incorporation into viral vectors, or suitable for in vitroprotein expression. The present invention further embodies the nucleicacid sequence of SEQ ID NO.: 1 in tandem with, or otherwise inconjunction with additional nucleic acids for the generation of fusionprotein products containing at least the functional segment of theprotein encoded for by the nucleic acid of SEQ ID NO.: 1. The presentinvention also encompasses the nucleic acid of SEQ ID NO.: 1 adapted foruse as a naked DNA transformant for incorporation and expression intarget cells. The present invention also provides for anti-sense DNAmolecule formulations which are the complement to nucleic acid sequenceof SEQ ID NO.: 1, and fragments thereof, whether complementary tocontiguous or discontinuous portions of the nucleic acid sequence of SEQID NO.: 1. The present invention also provides for compositionsincorporating modified nucleotides or backbone components which encodefor the nucleic acid sequence of SEQ ID NO.: 1, its complement, orfragments thereof. Such modified nucleotides and nucleic acids are knownin the art (see for example Verma et al., Ann. Rev. Biochem. 67: 99-134(1998)). Thus the present invention encompasses modified nucleic acidswhich incorporate, for example, intemucleotide linkage modification,base modifications, sugar modification, nonradioactive labels, nucleicacid cross-linking, and altered backbones including PNAs (polypeptidenucleic acids).

The present invention provides for the novel human checkpoint kinaseprotein hCDS1, which consists of the amino acid sequence of SEQ ID NO.:2. The invention encompasses hCDS1 protein produced by recombinant DNAtechnology and expressed in vivo or in vitro. The invention thusencompasses hCDS1 protein produced by transformed host cells insmall-scale or large-scale production. The invention encompassescomplete hCDS1 protein, in either glycosylated or unglycosylated forms,produced by either eukaryotic or prokaryotic cells. The presentinvention provides for hCDS1 protein expressed from mammalian, insect,plant, bacterial, fungal, or any other suitable host cell. The presentinvention encompasses hCDS1 protein that is produced as a fusion proteinproduct, conjugated to a solid support, or hCDS1 protein which islabeled with any chemical, radioactive, fluorescent, chemiluminescent orotherwise detectable marker. The present invention also provides forhCDS1 protein isolated from natural sources and enriched in purity overthat found in nature. The present invention also provides forpharmaceutical formulations of hCDS1 protein and formulations of thehCDS1 protein in pharmaceutically acceptable carriers or excipients.

The present invention encompasses any nucleic acid sequence which wouldencode for the amino acid sequence of SEQ ID NO.: 2, and the embodimentsof these nucleic acid sequences as described for SEQ ID NO.: 1, as thenucleic acid code for generating any nucleic acid sequence which willencode for a protein having the amino acid sequence of SEQ ID NO.: 2 ispredictable to one of skill in the art.

The present invention encompasses antibodies which bind specifically tothe hCDS1 protein, either polyclonal or monoclonal, as generated by theimmunization of a mammal with protein having the amino acid sequence ofSEQ ID NO.: 2, or fragments thereof.

The present invention also encompasses equivalent proteins wheresubstitutions of amino acids in the sequence of SEQ ID NO.: 2 that arereasonably predictable as being equivalent, and the embodiments thereofas described for SEQ ID NO.: 2. For example, non-polar (hydrophobicside-chain) amino acids alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, methionine; uncharged polar amino acidsglycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine;charged polar amino acids aspartic acid, glutamic acid; basic aminoacids lysine, arginine, and histidine are understood by those in the artto have functionally predictable effects when substituted. Thus thepresent invention also encompasses equivalent nucleic acids which encodefor such equivalent proteins and the embodinients thereof as describedfor SEQ ID NO.: 1.

The invention also provides for methods of generating hCDS1 protein, byusing recombinant DNA technology and the appropriate nucleic acidencoding for hCDS1 protein, fusion protein, or fragments thereof. Theinvention provides for incorporating an appropriate nucleic acidsequence into a suitable expression vector, the incorporation of anysuitable control elements such as promoter, enhancer, either inducibleor constitutively expressed. The invention provides for the use ofexpression vectors with or without at least one additional selectablemarker or expressible protein. The invention provides for methodswherein a suitably constructed expression vector is transformed orotherwise introduced into a suitable host cell, and protein is expressedby such a host cell. Thus the present invention also provides for thetransformed host cells, which are capable of producing hCDS1 protein,fusion protein, or fragments thereof.

The discovery that hCDS1 acts in coordination with Cdc25 in the DNAdamage checkpoint allows for the use of the compounds of the inventionin methods for therapeutic treatment of diseases which involve abnormalDNA damage checkpoint function. The present invention further providesfor the use of the compounds of the present invention as therapeuticsfor the treatment of cancer. In particular, the present invention allowsfor the specific modification of the hCDS1 -Cdc25 DNA damage checkpointin cells.

The present invention also encompasses methods for screening testcompounds for efficacy in effecting the hCDS1 mediated checkpointfunction of eukaryotic cells, said method comprising contacting a testcompound to eukaryotic cells, and detecting any change in hCDS1expression or function. Thus the invention further encompasses themethod of screening wherein said detection of change in hCDS1 expressionor function is accomplished by assaying for hCDS1 mRNA production, or byassaying for hCDS1 protein expression. In particular, the presentinvention allows for the screening of candidate substances for efficacyin modifying the DNA damage checkpoint by screening for any change inCdc25 phosphorylation, or kinase activity. The compounds or substancesidentified by the assays of the invention, or compounds corresponding tosuch compounds or substances, can be used for the manufacture ofpharmaceutical therapeutics.

Thus, in one embodiment the present invention provides forpharmaceutical compositions which include the hCDS1 protein, hCDS1nucleic acid, hCDS1 anti-sense nucleic acids. In another embodiment, thepresent invention provides for compounds or substances identified assuitable for use as a therapeutic by the assays of the invention, inpharmaceutical formulations. These pharmaceutical compositions canfurther include chemotherapeutic agents for the use in treating cancer,or be administered in a regimen coordinated with the administration ofother anti-cancer therapies. The present invention, in one embodimentthus encompasses methods for combined chemotherapy using the hCDS1derived pharmaceuticals independently, and in combination with otherchemotherapeutic agents, and in a second embodiment as admixtures withother anti-cancer therapeutics for single dose administration.

According to one aspect of the present invention, there is provided anucleic acid encoding hCDS1 protein having the amino acid sequenceillustrated in FIG. 2 (SEQ ID NO.: 2), or encoding a functionalequivalent fragment, or bioprecursor of said protein. Preferably, thenucleic acid may be a DNA molecule such as a genomic DNA molecule andeven more preferably a cDNA molecule, however it may also be RNA.

In a preferred embodiment, a nucleic acid encoding hCDS1 proteincomprises the nucleic acid sequence represented by position 66 to 1694of the sequence illustrated in FIG. 1 (SEQ ID NO.: 1), the complementthereof, or a nucleic acid sequence capable of hybridizing to eitherunder high stringency conditions.

The nucleic acid sequences defined herein may, advantageously, becapable of hybridizing under low stringency conditions to nucleic acidsequences derived from family members to identify homologs therefrom oralternatively to identify nucleic acid sequences from other species.

As would be well known to those skilled in the art due to the degeneracyof the genetic code the nucleic acid sequences according to theinvention may include substitutions therein yet which still encode thesame amino acid sequence.

Advantageously, the nucleic acids according to the invention may beincorporated into an expression vector and may be subsequently used totransforrn, transfect or infect a suitable host cell. In such anexpression vector the nucleic acid according to the invention isoperably linked to a control sequence, such as a suitable promoter orthe like, ensuring expression of the proteins according to the inventionin a suitable host cell. The expression vector may, advantageously be aplasmid, cosmid, virus or other suitable vector. The expression vectorand the host cell transfected, transformed or infected with the vectoralso form part of the present invention. Preferably, the host cell is aeukaryotic cell or a bacterial cell and even more preferably a mammaliancell or insect cell. Mammalian host cells are particularly advantageousbecause they provide the necessary post-translational modifications tothe expressed proteins according to the invention, such as glycosylationor the like, which modifications confer optimal biological activity onsaid proteins, which when isolated may advantageously be used indiagnostic kits or the like.

The expression vector including said nucleic acid according to theinvention may advantageously be used in vivo, such as in, for example,gene therapy.

According to a further aspect of the invention there is also provided atransgenic cell, tissue or organism comprising a transgene capable ofexpressing hCDS1 protein, which protein comprises the amino acidsequence illustrated in FIG. 2 (SEQ ID NO.: 2), or the amino acidsequence of a functional equivalent or bioprecursor or fragmenttherefor. The term “transgene capable of expression” as used hereinmeans a suitable nucleic acid sequence which leads to expression ofhCDS1 or proteins, having the same fumction and/or activity. Thetransgene may include, for example, genomic nucleic acid isolated fromhuman cells or synthetic nucleic acid, including DNA integrated into thegenome or in an extrachromosomal state. Preferably, the transgenecomprises the nucleic acid sequence encoding the proteins according tothe invention as described herein, or a functional fragment of saidnucleic acid. A functional fragment of said nucleic acid should be takento mean a fragment of the gene comprising said nucleic acid coding forthe proteins according to the invention or a functional equivalent,derivative or a non-functional derivative such as a dominant negativemutant, or bioprecursor of said proteins. For example, it would bereadily apparent to persons skilled in the art that nucleotidesubstitutions or deletions may be used using routine techniques, whichdo not affect the protein sequence encoded by said nucleic acid, orwhich encode a functional protein according to the invention.

The hCDS1 protein expressed by said transgenic cell, tissue or organismor a functional equivalent or bioprecursor of said protein also formspart of the present invention.

Further provided by the present invention is an antisense molecule whichis capable of hybridizing to the nucleic acid according to theinvention. Advantageously, the antisense molecule according to theinvention may be used as a medicament, or in the preparation of amedicament for the treatment of cancer and other proliferative diseases.

The present invention also advantageously provides nucleic acidsequences of at least approximately 15 nucleotides of a nucleic acidaccording to the invention and preferably from 15 to 50 nucleotides.These sequences may advantageously be used as probes or primers toinitiate replication, or the like. Such nucleic acid sequences may beproduced according to techniques well known in the art, such as byrecombinant or synthetic means. They may also be used in diagnostic kitsor the like for detecting the presence of a nucleic acid according tothe invention. These tests generally comprise contacting the probe withthe sample under hybridizing conditions and detecting for the presenceof any duplex or triplex formation between the probe and any nucleicacid in the sample.

Advantageously, the nucleic acid sequences, according to the inventionmay be produced using such recombinant or synthetic means, such as forexample using PCR cloning mechanisms which generally involve making apair of primers, which may be from approximately 15 to 50 nucleotidesspanning a region of the gene which is desired to be cloned, bringingthe primers into contact with mRNA, cDNA, or genomic DNA from a humancell, performing a polymerase chain reaction under conditions whichbring about amplification of the desired region (and where necessaryfirst performing a reverse transcription step), isolating the amplifiedregion or fragment and recovering the amplified DNA. Generally, suchtechniques as defined herein are well known in the art, such asdescribed in Sambrook et. al., (Molecular Cloninz; a Laboratory Manual,1989). Advantageously, human allelic variants of the nucleic acidaccording to the invention may be obtained by for example, probinggenomic DNA libraries from a range of individuals for example fromdifferent populations, and other genotyping techniques. Furthermore,nucleic acids and probes according to the invention may be used tosequence genomic DNA from patients, using techniques well known in theart, for example, the Sanger dideoxy chain termination method, which mayadvantageously ascertain any predisposition of a patient to certainproliferative disorders.

Further provided by the present invention are isolated proteins havingthe amino acid sequences as illustrated in FIG. 2 (SEQ ID NO.: 2) or theamino acid sequence of a functional equivalent functional fragment orbioprecursor of said protein in addition to antibodies, monoclonal orpolyclonal capable of binding to the amino acid sequences of theseproteins or fragments thereof. As would be well known to those skilledin the art, the proteins according to the invention may compriseconservative substitutions, deletions or insertions wherein the proteincomprises different amino acids than those disclosed in FIG. 2, yetwhich substitutions, deletions or insertions do not affect the activityof the proteins according to the invention or their ability to interactin the human cell cycle checkpoint pathway.

Preferred fragments include those comprising an epitope of the proteinsaccording to the invention. The epitopes may be determined using, forexample, peptide scanning techniques as described in Geysen et. al.,Mol. Immunol., 23; 709-715 (1986).

The antibodies according to the invention may be produced according totechniques which are known to those skilled in the art. Monoclonalantibodies may be prepared using conventional hybridoma technology asdescribed in Kohler F and Milstein C (1985), Nature 256, 495-497.Polyclonal antibodies may also be prepared using conventional technologywell known to those skilled in the art, and which comprises inoculatinga host animal, such as a mouse, with a protein or epitope according tothe invention and recovering the immune serum. The present inventionalso includes fragments of whole antibodies which maintain their bindingactivity, such as for example, Fv, F(ab′) and F(ab′)₂ fragments as wellas single chain antibodies.

Advantageously, the nucleic acid and/or the proteins according to theinvention may be included in a pharmaceutical composition together witha pharmaceutically acceptable carrier, diluent or excipient therefor.The pharmaceutical composition containing said nucleic acids accordingto the invention may, for example, be used in gene therapy. Such nucleicacids, according to the invention, may be administered naked, orpackaged in protein capsules, lipid capsules, liposomes, membrane basedcapsules, virus protein, whole virus, cell vectors, bacterial cellhosts, altered mammalian cell hosts, or such suitable means foradministration.

There is further provided by the present invention a method fordetecting for the presence or absence of a nucleic acid according to theinvention, in a biological sample, which method comprises, a) bringingsaid sample into contact with a probe comprising a nucleic acid or probeaccording to the invention under hybridizing conditions, and b)detecting for the presence of hybridization, for example, by thepresence of any duplex or triplex formation between said probe and anynucleic acid present in said sample. Proteins according to the inventionmay also be detected by a) contacting said sample with an antibody to anepitope of a protein according to the invention under conditions whichallow for the formation of an antibody-antigen complex, b) monitoringfor the presence of any antigen-antibody complex.

Kits for detecting said nucleic acids and proteins are also provided bythe present invention. A kit for detecting for the presence of a nucleicacid according to the invention in a biological sample may comprise (a)means for contacting the sample with a probe comprising a nucleic acidor a probe according to the invention and means for detecting for thepresence of any duplex or triplex formation between said probe and anynucleic acid present in the sample.

Likewise, a kit for detecting for the presence of a protein according tothe invention in a biological sample may comprise (a) means forcontacting said sample with an antibody to an epitope of a proteinaccording to the invention under conditions which allow for theformation of an antibody protein complex, and means for monitoring saidsample for the presence of any protein antibody complex.

A further aspect of the present invention provides a method ofdetermining whether a compound is an inhibitor or an activator ofexpression or activity of the proteins of the human cell cyclecheckpoint pathway which method comprises contacting a cell expressingthe proteins in said pathway with said compound and comparing the levelof expression of any of the proteins of the checkpoint pathway of saidcell against a cell which has not been contacted with said compound. Anycompounds identified may then advantageously be used as a medicament orin the preparation of a medicament for treating cancer or proliferativedisorders. Alternatively, the compounds may be included in apharmaceutical composition together with a pharmaceutically acceptablecarrier, diluent or excipient therefor. Advantageously, any compoundsidentified as an inhibitor of the cell checkpoint pathway may beincluded in a pharmaceutical composition according to the inventiontogether with a cytotoxic agent, such as a DNA damaging chemotherapeuticagent, and a pharmaceutically acceptable carrier diluent or excipienttherefor. Thus, the human cell cycle checkpoint inhibitor may enhancethe chemotherapeutic effect of cytotoxic agents used in, for example,anti-cancer therapy.

There is also provided by the present invention a method for screeningcandidate substances for anti-cancer therapy, which method comprises a)providing a protein according to the present invention exhibiting kinaseactivity together with a substrate for said protein under conditionssuch that the kinase will act upon the substrate, b) bringing theprotein and substrate into contact with a candidate substance, c)measuring the degree of any increase or decrease in the kinase activityof the protein, d) selecting a candidate substance which provides adecrease or increase in activity. Such a candidate substance may also beused as a medicament, or in the preparation of a medicament for thetreatment of cancer or other such proliferative cell disorders.

The present invention also comprises a method of identifying otherproteins active in the cell checkpoint pathway, which method comprisesa) contacting a cell extract with an antibody to an epitope of a proteinaccording to the invention, under appropriate binding conditions, b)identifying any antibody-protein complex and c) analyzing the complex toidentify any protein bound to the antibody or protein which is otherthan the protein according to the invention.

Another method for identifying proteins involved in the cell checkpointpathway utilizes a two-hybrid system developed in yeast by Chien et.al., supra (1991). This technique is based on functional in vivoreconstitution of a transcription factor which activates a reportergene. More particularly the technique comprises providing an appropriatehost cell with a DNA construct comprising a reporter gene under thecontrol of a promoter regulated by a transcription factor having a DNAbinding domain and an activating domain, expressing in the host cell afirst hybrid DNA sequence encoding a first fusion of a fragment or allof a nucleic acid sequence according to the invention and either saidDNA binding domain or the activating domain of the transcription factor,expressing in the host cell at least one second hybrid DNA sequenceencoding putative binding proteins to be investigated together with theDNA binding domain or activating domain of the transcription factorwhich is not incorporated in the first fusion; detecting any binding ofthe protein being investigated with a protein according to the inventionby detecting for the production of any reporter gene product in the hostcell; optionally isolating second hybrid DNA sequences encoding thebinding protein. In one embodiment of this aspect of the invention themethod may comprise:

(a) constructing at least two nucleotide vectors, the first of whichcomprises a nucleotide segment encoding for a DNA binding domain of GAL4protein operably linked to a nucleic acid sequence encoding a proteinaccording to the present invention, the second vector comprising anucleotide sequence encoding a protein binding domain of GAL4 operablylinked to a nucleotide sequence encoding a protein to be tested,

(b) co-transforming each of said vectors into a yeast cell beingdeficient for transcription of genes encoding galactose metabolizingproteins, wherein interaction between said test protein and the proteinaccording to the invention leads to transcription of galactose metabolicgenes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more clearly understood from the following exampleswhich are given by way of example only, with reference to theaccompanying drawings, wherein:

FIG. 1 illustrates the nucleotide sequence of hCDS1 (SEQ ID NO.: 1)wherein residues 66-1694 is the coding region, and 3′ and 5′ depicts theuntranslated regions(UTRs). The initiation and termination codons areshown in bold,

FIG. 2 illustrates the deduced amino acid sequence of hCDS1 (SEQ ID NO.:2),

FIG. 3 illustrates the amino acid sequence alignment of hCDS1 and S.pombe cds1 performed using the CLUSTAL W alignment program and annotatedusing the GENEDOC program. Residues shaded black are identical betweenthe two proteins and residues shaded grey are similar.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention encompasses the isolation and characterization ofa novel human checkpoint kinase gene and protein which is called hCDS1.The hCDS1 gene and protein show some similarity to a homologous gene andprotein found in S. pombe.

The S. pombe cds1+ gene was identified by its ability to complement aDNA polymerase a mutant (Murakami & Okayama, 1995, Nature, 374:817-819). S. pombe cds1 was also able to suppress the hydroxyureasensitivity (DNA replication-dependent checkpoint) of rad1, rad3 andrad9 mutant S. pombe strains but not the UV sensitivity (DNAdamage-dependent checkpoint). This shows that S. pombe cds1 executes itscheckpoint function during DNA synthesis. S. pombe cds1 is a putativeprotein kinase that is 70% similar to the S. cerevisiae checkpoint geneRAD53. In S. cerevisiae the DNA damage and DNA replication-dependentcheckpoints are genetically separate at the level of detection of DNAlesions. The two pathways then converge on the Rad53 protein kinasewhich potentially acts as an amplifier in the signal transductionpathway. This appears not to be the case in S. pombe where the sameproteins are involved in detection of all types of lesion but thetransduction of the signal follows separate pathways involving differentprotein kinases; S. pombe cds1 for the DNA replication-dependentcheckpoint and Chk1/Rad27 for the DNA damage-dependent pathway. It hasbeen suggested that S-phase-specific activation of cds1 kinase maydefine a subpathway of the checkpoint response in S. pombe (Lindsay etal., 1998, Genes and Development, 12: 382-395).

S. pombe cds1 may act via an interaction with DNA polymerase a tomonitor the progress of DNA replication or the integrity of replicationcomplexes. There is evidence in Drosophila for a kinase of theappropriate molecular weight associating with DNA polymerase α (Peck etal., 1993, B.B.R.C., 190: 325-331). Alternatively it may act viaphosphorylation of p107^(weel) in a manner analogous to Chk1 ultimatelyaffecting the activity of the G1/S phase cyclin dependent kinases.

Many of the methods and materials for carrying out the basic molecularbiology manipulations as described in the examples below are known inthe art, and can be found in such references as Sambrook et al.Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press(1989); Berger et al., Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc., (1987); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing Co., Inc.(1986); Ausubel et al., Short Protocols in Molecular Biology, 2nd ed.,John Wiley & Sons, (1992); Goeddel Gene Expression Technology, Methodsin Enzymology, Vol. 185, Academic Press, Inc., (1991); Guthrie et al.,Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology,Vol. 194, Academic Press, Inc., (1991); McPherson et al., PCR Volume 1,Oxford University Press, (1991).

The invention in its several aspects can be more readily understood byreviewing the following examples.

EXAMPLE 1 Isolation of hCDS1

Isolation of hCDS1 began with a search for sequences similar to S. pombecds1+ using the TBLASTN program. A human expressed sequence tag (EST No.864164) was identified in the proprietary LifeSeqR) database (IncytePharmaceuticals Inc., Palo Alto, Calif., USA). Sequence analysis of the1.3 kb insert revealed an incomplete open reading frame which wassimilar to S. pombe cds1. Approximately 650 nucleotides of novel 5′ DNAsequence was obtained by 5′RACE (rapid amplification of cDNA ends) usinga Marathon Ready human placental cDNA (Clontech), following themanufacturer's instructions.

Briefly, the two hCDS1 gene specific primers used for nested PCR(Polymerase chain reaction) reactions were GSP35′-TTTTGCTGATGATCTTTATGGCTAC-3′ (SEQ ID NO.: 3) and GSP45′-CACAGGCACCACTTCCAAGAGTTTT-3′ (SEQ ID NO.: 4). Subsequently. acomplete ORF for hCDS1 was amplified from a human SK-N-MC neuroblastomacDNA library using the PCR primers5′-GGGCTCGAGAGCAGCGATGTCTCGGGAGTCGGATGT-3′ (SEQ ID NO.: 5) and5′-GGCGGATCCTCGAGTCACAACACAGCAGCACACAC-3′ (SEQ ID NO.: 6). Theamplification product was then cloned into pCR2.1 vector (Invitrogen)and the DNA sequence determined.

The nucleic acid sequence of hCDS1 was found to show 47.8% identity tothe S. pombe cds1+ at the DNA level. Termination codons were present inall three reading frames in the 120 nucleotides immediately 5′ to theputative hCDS1 initiation codon, indicating that the complete codingregion has been isolated. Parts of the sequence were also found to matchpartial sequences found in the NCBI databases, EST AA285249, genomicsequence H55451, and the 54 base pair fragment H55698.

The identified human gene and vectors encoding the hDCS1 nucleic acidsequence were deposited on Oct. 6, 1997 as plasmid HCDS1 ORF/pCR-Bluntdeposited under Accession No. LMBP 3708; plasmid HCDS1 5′RACEfragment/pGEM-Easy deposited under Accession No. LMBP 3710; and plasmidHCDS1 3′fragment Incyte clone 864164/pSPORT deposited under AccessionNo. LMBP 3709 with the Belgian Co-ordinated Collections ofMicro-organisms (BCCM) at Laboratorium Voor MoleculaireBiologies-Plasmidencollecte (LMBP) 35, B-9000 Gent, Belgium, inaccordance with the provisions of the Budapest Treaty, Apr. 28, 1997.

The tissue expression profile of hCDS1 was examined on multiple tissueNorthern blots (Clontech) and a cancer cell line Northern blot(Clontech), which were probed with the hCDS1 ORF. A single transcript ofapproximately 2.1 kb was observed. Expression was undetectable byconventional Northern blot hybridization conditions in all normal humantissues examined. However, expression was found to be greatly elevatedin all of the cancer cell lines examined.

The hCDS1 gene was localized to chromosome 22q11.2-q12, as determinedusing the complete ORF as a probe for FISH (Fluorescent in situHybridization) analysis. The hybridization efficiency was approximately62%, and no other loci were detected under the conditions used.

Briefly, lymphocytes isolated from human blood were cultured ina-minimal essential media (MEM) supplemented with 10% fetal calf serumand phytohaemagglutinin (PHA) at 37° C. for 68-72 hours. The lymphocytecultures were treated with BrdU (0.18 mg/ml, Sigma) to synchronize thecell population. The synchronized cells were washed three times withserum-free medium to release the block and re-cultured at 37° C. for 6hours in α-MEM with thymidine (2.5 μg/ml Sigma). Cells were harvestedand slides were prepared using standard procedures including hypotonictreatment, fixation and air-drying. DNA fragments containing the hCDS 1complete ORF were gel purified and biotinylated with dATP using the BRLBioNick labeling kit (15° C. 1 hour) (Heng et al., 1992, Proc. Natl.Acad. Sci. USA. 89: 9509-9513).

Slides were then baked at 55° C. for 1 hour, and after RNase treatment,the slides were denatured in 70% formamide in 2×SSC for 2 minutes at 70°C. followed by dehydration with ethanol. Probes were denatured at 75° C.for 5 minutes in a hybridization mix consisting of 50% formamide and 10%dextran sulphate. Probes were loaded on the denatured chromosomalslides. After overnight hybridization, slides were washed and detected.FISH signals and the DAPI-banding pattern were recorded separately bytaking photographs, and the assignment of the FISH mapping data withchromosomal bands was achieved by superimposing FISH signals with DAPIbanded chromosomes (Heng & Tsui, 1994, Methods in Mol. Biol., 33:35-49).

EXAMPLE 2 Characterization of hCDS1 protein

The hCDS1 nucleic acid sequence cDNA predicts a translation product of543 amino acids with an approximate molecular weight of 61kDa. This isclose to the apparent molecular weight of endogenous Cds1 protein inHeLa cells. The predicted hCDS1 protein, is 28% identical to the cds1protein of S. pombe, 28% identical to RAD53 and 27% identical to theDUN1 kinase of S. cerevisiae. Sequence alignment of these apparenthomologs shows several regions of sequence similarity outside the kinasedomain, including conservation of the Fork Head Associated domain(Hoffmann et al., 1995, Trends Biochem. Sci., 20: 347-9). The humanprotein shows the same overall structure as S. pombe CDS1 and Scerevisiae DUN1 in that it lacks the long C-terminal extension found inRAD53. Northern blot analysis with hCDS1 identified a single transcriptof about 2.2 kb expressed in testis and in 8 human cancer samplesexamined.

Briefly, two multiple tissue Northern blots (Clontech) and a Cancer Cellline Northern blot (Clontech) were hybridized with a cDNA probe forhCDS1. The probe corresponds to the complete ORF as described above. Theblots were washed at high stringency (0.1×SSC, 0.1% SDS, 50° C., 2×20min) and exposed using Kodak X-OMAT autoradiography film withintensifying screens at −70° C.

EXAMPLE 3 Cdc25 Total Activity Assay

The possibility that dephosphorylation of Cdc2 is down-regulated in thepresence of DNA damage required an assay to allow for the analysis ofthe total activity of Cdc25. In the presence of EDTA, Cdc2/Cyclin B fromasynchronous HeLa cell extracts was found to inactivate spontaneously.

Briefly, cells were lysed in ice-cold lysis buffer (50 mM Tris pH 7.4containing 2 mM magnesium chloride, 1 mM phenylmethylsulphonyl fluoride,and 5 μg/ml leupeptin, pepstatin and aprotinin). Lysates were cleared bycentrifugation at 10,000×g for 10 minutes and the protein concentrationof the supernatants determined using the Lowry assay. 10 mM EDTA wasadded to the supernatants (100 μg in 60 μL) and the reaction initiatedby incubation at 30° C.At assay intervals the activity of Cdc2/Cyclin Bwas assayed by measuring the histone-H1 kinase activity present inanti-Cyclin B irmmune-precipitates (Blasina et al., supra.). Forinununoblots 400 μg of cell lysate was immune-precipitated usinganti-Cyclin B antibody, resolved on an 11% acrylamide-SDS gel.Monoclonal antibody against the PSTAIRE motif of Cdc2 was used to detectthe different phospho-forms of Cdc2.

Activation correlates with loss of the inhibited-phosphorylated form ofCdc2, visualized as the slower migrating species on SDS-PAGE gels.Activation was prevented by vanadate, an inhibitor of Cdc25 and othertyrosine phosphatase. Furthermore, imnmune-depletion withCdc25C-specific anti-sera greatly reduced activation of Cdc2/Cyclin B.There was no increase in the levels of Cdc2 or Cyclin B protein,phosphorylation by WEE1 and Myt1 was blocked by the presence of 10 mMEDTA. Thus, these result demonstrate that the activation of Cdc2 was theresult of dephosphorylation. In lysates of asynchronous HeLa cells, theendogenous Cdc25 phosphatase activity is sufficient to dephosphorylateand activate more than 80% of the available Cyclin B/Cdc2 in 30 minutes.Analysis of lysates of HeLa cells in which the DNA had been damaged byexposure to 10 Gy of τ-irradiation one hour before harvesting showed asignificant reduction in the rate of activation of Cdc2, such that isless than 25% of the available Cdc2/Cyclin B was activated during the 30rninutes incubation. The amount of Cdc2/Cyclin B in complex was notsignificantly altered and it was activated to the same extent as controlCdc2/Cyclin B by addition of exogenous GST-Cdc25. Irradiation with 10 Gyled to more than 3-fold reduction in the rate of Cdc2 dephosphorylationin the 10 time courses examined. If the inactivation of Cdc25 measuredabove is part of the DNA-damage checkpoint response in human cells, thenexperimental conditions that over-ride the DNA damage checkpoint mightbe expected to block the radiation-induced inhibition of Cdc25.

EXAMPLE 4 DNA Damage Checkpoint Effect of hCDS1

DNA damage response in a variety of cells is known to require variousrelated kinases which structurally are related to PI-3 kinases. At leastone member of the family, DNA-Protein Kinase, has been shown to besensitive to wortmannin in vitro (Hawley et al., 1996, Genes and Dev.10: 2383-8; Hartley et al., 1995, Cell. 82: 849-856). Thus thepossibility that a wortmannin-sensitive kinase acted upstream of theradiation induced delay in M-phase entry was tested (Price et al., 1996,Cancer Research, 56: 246-250). HeLa cells can be arrested in M-phase bynocodazole, irradiation causes cells to delay in G2 prior to thenocodazole-sensitive M-phase block point. Thus, by scoring the mitoticindex of cells that are cultured in nocodazole, it is possible todetermine whether entry into mitosis has been delayed. Control cellscultured in the presence of nocodazole for 14 hours contained 60%mitotic cells, the presence of wortmannin had little effect on thisnumber. However, irradiation reduced the number of cells that reach thenocodazole block point to only 10%. In contrast, irradiation in thepresence of wortmannin had only a modest effect. These resultsdemonstrate that wortmanniniover-rides the DNA damage G2 checkpoint inHeLa cells.

The effects of wortmannin on the radiation-induced inactivation of Cdc25was then tested. Wortmannin had little effect on the activation ofCdc2/Cyclin B in extracts prepared from unirradiated cultures, howeverit did greatly diminish the irradiation-induced decrease in Cdc25activity.

Radiation-induced G2 checkpoint is also over-ridden in cell-linesderived from ad patients with the genetic disorder ataxiatelangiectasia. Ataxia Telangiectasia mutant cells (ATM) are defectivein both the G1 and G2 checkpoints following exposure to many, but notall, agents that damage DNA (Canman et al., 1994, Cancer Research 54:5054-5058). The failure of AT-deficient cells to delay G1 correlateswith a failure to up-regulate p53 (Kastan et al., 1992, Cell, 71:587-589), and with failure to phosphornlate and activate cAb1 (Baskaranet al., 1997, Nature, 387: 516-519; Shafman et al., 1997, Nature. 387:520-523). The molecular basis for the failure to delay G2 is unknown.AT-deficient cells show greatly reduced responses to agents thatgenerate chromosomal breaks, such as ionizing τ-rays. Remarkably,AT-deficient cells have near normal responses following the base damagethat is generated by irradiation with a UV source (Canman et al., 1994,Cancer Research. 54: 5054-5058; Painter et al., 1980, Proc. Natl. Acad.Sci. USA. 77: 7315-7317; Zamnpetti-Bosseler et al., 1981, Int. J.Radiat. Biol., 39: 547-558). The effects of UV and T-irradiation on theCdc25 activity of AT-plus and AT-minus SV40-transformed human fibroblastcell-lines was tested. AT-minus cells respond to UV-irradiation with arobust reduction in the rate at which Cdc2 is dephosphorylated. Incontrast, T-irradiation had only a modest effect on the rate ofdephosphorylation of Cdc2. In AT-plus cells the rate ofdephosphorylation of Cdc2 was significantly reduced following eitherionizing-radiation or UV-radiation. These data indicate that the ATMgene product is required for the efficient inactivation of Cdc25following r-irradiation and demonstrate a correlation betweeninactivation of Cdc25 and delayed entry into M-phase following DNAdamage.

Mediators of the checkpoint-dependent inactivation of Cdc25 in humancells are excellent targets for generating therapeutics or therapeuticregimens that will enhance anti-cancer treatment, and reduceside-effects on normal cells.

To facilitate biochemical characterization of hCDS1, 6his-hCDS1 wasexpressed in insect cells, affinity purified and incubated in extractsof HeLa cells in the presence of an ATP-regenerating system. EDTA wasadded to inhibit kinase in the extract, and the rate ofdephosphorylation and activation of Cdc2/CyclinB was monitored.

Briefly, recombinant viruses encoding for 6his-hCDS1, 6his-Chk1,6his-Cdc2 and GST-Cdc25C were generated using the Bac-to-Bac expressionsystem from Gibco/BRL. 6his-fusion proteins were purified following theprocedure described in Kumagai et al., (1995), Mol. Biol. Cell. 6:199-213. GSH-sepharose beads were incubated for 15 minutes in Sf9extracts; beads were collected by centrifugation and washed three-timeswith lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1% NP40,5% glycerol, 0.1% β-mercaptoethanol and protease inhibitors). Beads werewashed three-times with kinase assay buffer (50 mM Tris pH 7.4, 10 mMMgCl₂) prior to phosphorylation reactions or three-times withphosphatase assay buffer (50 mM imidazole pH 7.4, 5 mM EDTA and 0.1%β-mercaptoethanol) prior to phosphatase assays.

Both 6his-Chk1 and 6his-hCDS1 were found to significantly reduce theactivation of Cdc2/Cyclin B in these assays. The reduced activation ofCdc2 was dose dependent and required ATP. Confirmation that Cdc2 was notirreversibly inhibited by 6his-Chk1 or 6his-hCDS1 was shown by theactivation that resulted when excess GST-Cdc25C was added after kinasetreatment. Thus, both 6his-hCDS 1 and 6his-Chk1 can mimic theradiation-induced down-regulation of Cdc25 seen in extracts. Theseexperiments used HeLa cell lysates that had been clarified bycentrifugation, therefore it is unlikely that changes in sub-cellularlocale could account for inactivation of Cdc25 (Peng et al., 1997,Science, 277: 1501-1505).

EXAMPLE 5 Direct Effect of hCDS1 on Cdc25

Indirect mechanisms of inhibition of Cdc25 by hCDS1 could not beexcluded by the cell lysate assays, therefore, affinity-purifiedreagents were used to determine direct phosphorylation and inhibition ofGST-Cdc25 activity by hCDS1.

GST-Cdc25 was incubated with either 6his-hCDS1, mock beads, or 6his-Chk1in the presence of τ-32P ATP for 15 minutes at 30° C. Proteins wereresolved by SDS-PAGE and visualized by autoradiography. GST-Cdc25 wasphosphorylated by 6his-Chk1 and by 6his-hCDS1. Assays were performed todetermine if Cdc25 phosphatase activity was effected by thisphosphorylation.

GST-Cdc25 was assayed for its ability to activate the histone-H1kitaseg-activity of Cdc2/Cyclin B irnmune-precipitates. It was foundthat phosphorylation of GST-Cdc25 by 6his-hCDS1 inhibited the ability ofGST-Cdc25 to activate Cdc2/Cyclin B. Thus, these data demonstrate that6his-hCDS1 inactivated Cdc25 in vitro, and that Cdc25 is inactivated invivo following DNA damage.

Since 6his-Chk1 associates with GST-Cdc25 and has histone-H1 kinaseactivity in vitro (Sanchez et al., 1997. Science, 277: 1497-1501),analysis of Cdc2/Cyclin B kinase activity was obscured. In order to testGST-Chk1 effects, an assay was used in which Cdc2 dephosphorylation wasmonitored by the disappearance of the slower migrating species of Cdc2on gel-mobility analysis.

Briefly, phosphorylated Cdc2 was purified from Sf9 cells that had beensimultaneously infected with recombinant baculoviruses encoding6his-Cdc2, 6his-Weel, 6his-Myt1 and GST-Cyclin B (Parker et al., 1992,Science, 257: 1955-1957. The 6his-Cdc2 complexed to Cyclin B waspurified using GSH beads under the conditions for GST-Cdc25 except that1 mM VO₄ was included in the lysis buffer. Western Blot analysis showedthat quadruple infection resulted in phosphorylation of the majority ofCdc2/GST-Cvclin B at one or both inhibitory sites. These phosphataseassays were carried out in the presence of 10 mM EDTA, and the absenceof ATP, conditions that eliminate the possibility of 6his-Chk1phosphorylating Cdc2 or Cyclin B directly. GST-Cdc25 catalyses areduction in the slower migrating phosphorylated forms of Cdc2. Priorphosphorylation of GST-Cdc25 by 6his-Chk1 leads to a dosedependentreduction in GST-Cdc25 activity. These data confirm that Chk1 negativelyregulated Cdc25 activity (Furnari et al., 1997, Science, 277: 1495-1497;Weinert, 1997, Science, 277: 1450), and extend them by demonstratingthat the negative regulation involves inactivation of the phosphataseactivity.

EXAMPLE 6 DNA Damage and Modification of hCDS1

As the previous data had shown that 6his-hCDS1 inactivates Cdc25, andthat DNA damage is associated with Cdc25 inactivation, an assay wasperformed to determine if DNA damage leads to any modification oractivation of hCDS1. Antisera raised against 6his-hCDS1 was used inimmune-complex kinase assays using HeLa cell lysates. A weak signalcorresponding to hCDS1 was detected in the sample from asynchronous HeLacells; increased phosphorylation of hCDS1 was seen followingirradiation.

Briefly, antibodies to hCDS1 were generated by immunizing a rabbit with6his-hCDS1 purified from Sf9 cells (Harlow et al., Antibodies (ColdSpring Harbor Laboratory Press. NY, 1988). The resulting antiseraimmune-precipitates an active kinase of the expected molecular weightfrom Sf9 cells infected with 6his-hDCS1 virus, but not from uninfectedSf9 cells, or from other cells infected with 6his-Chk1 virus.

The results were confirmed as being due to hDCS1 by re-precipitation ofthe protein band following denaturation in 4% SDS. The in vitrophosphorylation is most likely due to autophosphorvlation, and theincreased signal reflects an increase in activity following irradiation.The increase of in vitro phosphorylation of p64^(cds1) suggests that,like RAD53 and DUN1, hCDS1 is modified in response to DNA damage.

The effect of arresting DNA synthesis on phosphorylation of p64^(cds1)was examined by further assay. The hCDS1 from replication arrested cellsbehaved exactly like the protein from asynchronous cultures; nosignificant increase in phosphorylation was seen in response tothymidine or other agents that block DNA replication. The increasedphosphorylation of p64^(cds1) was detected following irradiation ofthyrmidine arrested cells. The effect of damaging DNA in cells that arepredominantly arrested outside S-phase was also tested. Cells werecultured in the presence of nocodazole for 20 hours prior toirradiation. Again, a weak but detectable signal was seen in theunirradiated sample. However, irradiation of nocodazole arrested cellsled to increased phosphorylation.

These findings surprisingly contrast with the results found in yeast,where fission yeast Cds1 has been found to be activated in response toincompletely replicated DNA (Boddy et al., 1998, Science, 280: 909-12;Lindsay et al., 1998, Genes and Dev., 12: 382-95). The results here showa role for human Cds1 in the

DNA damage checkpoint rather than the replication checkpoint aspreviously found in yeast.

EXAMPLE 7 Drug Identification

The Cdc25 assays described above are suitable for use in theidentification of chemical agents that would modify the DNA damagecheckpoint mediated by hCDS1 and Cdc25, either by enhanced or inhibitedactivity. Thus a typical screening assay would use similar conditions asdescribed above, plus addition of a reagent to be tested. Monitoring ofthe activity of the assay components, i.e. detection of phosphorylationas described above, can be conducted in comparison to control reactionsto detect both enhanced and inhibited activity.

Clearly such assays are readily adaptable to mechanical/automatedapparatus and detection. With the fundamental elements of the assayreactions being known, the assay is clearly suited for use inconjunction with automated high-throughput low-signal apparatus whichmay incorporate microscopic slide arrays, or cell-biochip arrays inconjunction with CCD detection devices and the use of a visible signaltriggered by phosphorylation or other reaction to kinase activity.

EXAMPLE 8 Therapeutic Use

The characterization of hCDS1 and the elucidation that the role forhuman Cds1 is in the DNA damage checkpoint rather than the replicationcheckpoint as found in yeast, allows for the adaptation of thisknowledge to the preparation of pharmaceuticals, and therapeutic methodsfor acting as an adjunct to chemotherapy of cancer.

In particular, pharmaceutical formulations of the present inventionincorporating cDNA, RNA, antisense molecules, hCDS1 protein, antibodiesagainst hCDS 1 protein, or other therapeutics corresponding to thoseidentified in the assays of the invention, can be administered inconjunction with any suitable chemotherapy agent in order to act as anadjunct to the main action of the chemotherapy agent. For example, theuse of anticancer drugs such as antimetabolite, antibiotics, alkylatingagents, microtubule inhibitors, steroid hormones and their antagonists,and others, is generally directed against metabolic sites essential tocell replication. While ideally these drugs should intervene only withthe cellular processes unique to malignant cells, the currentlyavailable anticancer drugs affect all proliferating cells, both normaland malignant. Thus, current chemotherapy is hampered by a steepdose-response curve for both toxic and therapeutic effects. Therefore,co-administration of the hDCS1-based drugs of the present invention, anddrugs identified by the hCDS1 assays of the present invention, withchemotherapeutic agents will allow for enhanced killing of malignantcells.

One mechanism for enhanced killing is effected by disabling the DNAdamage checkpoint control of malignant cells, thus making theadministration of DNA damaging chemotherapeutic agents more effective.The disabling of the DNA damage control checkpoint can be effected bymodifying the hCDS1 response, as demonstrated by the data above.

Thus, the co-administration of novel hCDS1 based therapeutics incombination with any one or more anticancer agent is contemplated by thepresent invention. For example. normal dosages of the anticancer drugsCytarabine, Fludarabine, 5-Fluorouracil, 6-Mercaptopurine, Methotrexate,6-Thioguanine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,Idarubicin, Plicarnycin, Carmustine, lomustine. Cyclophosphamide,Ifosfamide, Mechloroethamine, Streptozotocin, Navelbine, Paclitaxel,Vinblastine, Vincristine, Asparaginase, Cisplatin, Carboplatin,Etoposide, Interferons, Procarbazine etc., can be administered with theappropriate amount of hDCS1 based drug so as to a) alter the length oftime of administration, b) alter the time between administrations, c)alter the efficacy of the chemotherapeutic agent on malignant cells, ord) alter the side-effects of the chemotherapeutic agent on normal cells.The effects of the co-administration of hCDS1 based drugs can be any oneor combination of these effects in addition to others.

Typically, destruction of cancer cells by chemotherapeutic agentsfollows first-order kinetics, for a log kill effect. Thus, theco-administration of hCDS1-based therapeutics would be designed toenhance the log kill effect. Typically, chemotherapeutic treatmentprotocols call for a combination of drugs which act at different stepsin the metabolic pathway, thus enhancing killing while staying belowtoxic levels. Thus, the co-administration of hCDS1 based therapeuticswould ideally be in combination with such protocols, and improveefficacy thereof.

Ultimately, the most effective therapeutic methods would combinetargeted administration of chemotherapeutic drugs andlor MDR (multidrugresistance) inhibiting agents, with hCDS1 based therapeutics, tospecifically target and eliminate malignant cells via the cells′ ownuncontrolled replication without DNA damage repair, and thus eventualcell death.

The foregoing discussion and examples are intended as illustrative ofthe present invention and are not to be taken as limiting. Still othervariants within the spirit and scope of this-invention are possible andwill readily present themselves to those of skill in the art.

6 1 1858 DNA Homo sapiens CDS (66)..(1694) 1 actagtgatt actcacagggctcgagcggc cgcccgggca ggtcaggtgg gctcacgcgg 60 tcgtg atg tct cgg gag tcggat gtt gag gct cag cag tct cat ggc agc 110 Met Ser Arg Glu Ser Asp ValGlu Ala Gln Gln Ser His Gly Ser 1 5 10 15 agt gcc tgt tca cag ccc catggc agc gtt acc cag tcc caa ggc tcc 158 Ser Ala Cys Ser Gln Pro His GlySer Val Thr Gln Ser Gln Gly Ser 20 25 30 tcc tca cag tcc cag ggc ata tccagc tcc tct acc agc acg atg cca 206 Ser Ser Gln Ser Gln Gly Ile Ser SerSer Ser Thr Ser Thr Met Pro 35 40 45 aac tcc agc cag tcc tct cac tcc agctct ggg aca ctg agc tcc tta 254 Asn Ser Ser Gln Ser Ser His Ser Ser SerGly Thr Leu Ser Ser Leu 50 55 60 gag aca gtg tcc act cag gaa ctc tat tctatt cct gag gac caa gaa 302 Glu Thr Val Ser Thr Gln Glu Leu Tyr Ser IlePro Glu Asp Gln Glu 65 70 75 cct gag gac caa gaa cct gag gag cct acc cctgcc ccc tgg gct cga 350 Pro Glu Asp Gln Glu Pro Glu Glu Pro Thr Pro AlaPro Trp Ala Arg 80 85 90 95 tta tgg gcc ctt cag gat gga ttt gcc aat cttgaa tgt gtg aat gac 398 Leu Trp Ala Leu Gln Asp Gly Phe Ala Asn Leu GluCys Val Asn Asp 100 105 110 aac tac tgg ttt ggg agg gac aaa agc tgt gaatat tgc ttt gat gaa 446 Asn Tyr Trp Phe Gly Arg Asp Lys Ser Cys Glu TyrCys Phe Asp Glu 115 120 125 cca ctg ctg aaa aga aca gat aaa tac cga acatac agc aag aaa cac 494 Pro Leu Leu Lys Arg Thr Asp Lys Tyr Arg Thr TyrSer Lys Lys His 130 135 140 ttt cgg att ttc agg gaa gtg ggt cct aaa aactct tac att gca tac 542 Phe Arg Ile Phe Arg Glu Val Gly Pro Lys Asn SerTyr Ile Ala Tyr 145 150 155 ata gaa gat cac agt ggc aat gga acc ttt gtaaat aca gag ctt gta 590 Ile Glu Asp His Ser Gly Asn Gly Thr Phe Val AsnThr Glu Leu Val 160 165 170 175 ggg aaa gga aaa cgc cgt cct ttg aat aacaat tct gaa att gca ctg 638 Gly Lys Gly Lys Arg Arg Pro Leu Asn Asn AsnSer Glu Ile Ala Leu 180 185 190 tca cta agc aga aat aaa gtt ttt gtc tttttt gat ctg act gta gat 686 Ser Leu Ser Arg Asn Lys Val Phe Val Phe PheAsp Leu Thr Val Asp 195 200 205 gat cag tca gtt tat cct aag gca tta agagat gaa tac atc atg tca 734 Asp Gln Ser Val Tyr Pro Lys Ala Leu Arg AspGlu Tyr Ile Met Ser 210 215 220 aaa act ctt gga agt ggt gcc tgt gga gaggta aag ctg gct ttc gag 782 Lys Thr Leu Gly Ser Gly Ala Cys Gly Glu ValLys Leu Ala Phe Glu 225 230 235 agg aaa aca tgt aag aaa gta gcc ata aagatc atc agc aaa agg aag 830 Arg Lys Thr Cys Lys Lys Val Ala Ile Lys IleIle Ser Lys Arg Lys 240 245 250 255 ttt gct att ggt tca gca aga gag gcagac cca gct ctc aat gtt gaa 878 Phe Ala Ile Gly Ser Ala Arg Glu Ala AspPro Ala Leu Asn Val Glu 260 265 270 aca gaa ata gaa att ttg aaa aag ctaaat cat cct tgc atc atc aag 926 Thr Glu Ile Glu Ile Leu Lys Lys Leu AsnHis Pro Cys Ile Ile Lys 275 280 285 att aaa aac ttt ttt gat gca gaa gattat tat att gtt ttg gaa ttg 974 Ile Lys Asn Phe Phe Asp Ala Glu Asp TyrTyr Ile Val Leu Glu Leu 290 295 300 atg gaa ggg gga gag ctg ttt gac aaagtg gtg ggg aat aaa cgc ctg 1022 Met Glu Gly Gly Glu Leu Phe Asp Lys ValVal Gly Asn Lys Arg Leu 305 310 315 aaa gaa gct acc tgc aag ctc tat ttttac cag atg ctc ttg gct gtg 1070 Lys Glu Ala Thr Cys Lys Leu Tyr Phe TyrGln Met Leu Leu Ala Val 320 325 330 335 cag tac ctt cat gaa aac ggt attata cac cgt gac tta aag cca gag 1118 Gln Tyr Leu His Glu Asn Gly Ile IleHis Arg Asp Leu Lys Pro Glu 340 345 350 aat gtt tta ctg tca tct caa gaagag gac tgt ctt ata aag att act 1166 Asn Val Leu Leu Ser Ser Gln Glu GluAsp Cys Leu Ile Lys Ile Thr 355 360 365 gat ttt ggg cac tcc aag att ttggga gag acc tct ctc atg aga acc 1214 Asp Phe Gly His Ser Lys Ile Leu GlyGlu Thr Ser Leu Met Arg Thr 370 375 380 tta tgt gga acc ccc acc tac ttggcg cct gaa gtt ctt gtt tct gtt 1262 Leu Cys Gly Thr Pro Thr Tyr Leu AlaPro Glu Val Leu Val Ser Val 385 390 395 ggg act gct ggg tat aac cgt gctgtg gac tgc tgg agt tta gga gtt 1310 Gly Thr Ala Gly Tyr Asn Arg Ala ValAsp Cys Trp Ser Leu Gly Val 400 405 410 415 att ctt ttt atc tgc ctt agtggg tat cca cct ttc tct gag cat agg 1358 Ile Leu Phe Ile Cys Leu Ser GlyTyr Pro Pro Phe Ser Glu His Arg 420 425 430 act caa gtg tca ctg aag gatcag atc acc agt gga aaa tac aac ttc 1406 Thr Gln Val Ser Leu Lys Asp GlnIle Thr Ser Gly Lys Tyr Asn Phe 435 440 445 att cct gaa gtc tgg gca gaagtc tca gag aaa gct ctg gac ctt gtc 1454 Ile Pro Glu Val Trp Ala Glu ValSer Glu Lys Ala Leu Asp Leu Val 450 455 460 aag aag ttg ttg gta gtg gatcca aag gca cgt ttt acg aca gaa gaa 1502 Lys Lys Leu Leu Val Val Asp ProLys Ala Arg Phe Thr Thr Glu Glu 465 470 475 gcc tta aga cac ccg tgg cttcag gat gaa gac atg aag aga aag ttt 1550 Ala Leu Arg His Pro Trp Leu GlnAsp Glu Asp Met Lys Arg Lys Phe 480 485 490 495 caa gat ctt ctg tct gaggaa aat gaa tcc aca gct cta ccc cag gtt 1598 Gln Asp Leu Leu Ser Glu GluAsn Glu Ser Thr Ala Leu Pro Gln Val 500 505 510 cta gcc cag cct tct actagt cga aag cgg ccc cgt gaa ggg gaa gcc 1646 Leu Ala Gln Pro Ser Thr SerArg Lys Arg Pro Arg Glu Gly Glu Ala 515 520 525 gag ggt gcc gag acc acaaag cgc cca gct gtg tgt gct gct gtg ttg 1694 Glu Gly Ala Glu Thr Thr LysArg Pro Ala Val Cys Ala Ala Val Leu 530 535 540 tgaactccgt ggtttgaacacgaaagaaat gtaccttctt tcactctgtc atctttcttt 1754 tctttgagtc tgtttttttatagtttgtat tttaattatg ggaataattg ctttttcaca 1814 gtcactgatg tacaattaaaaacctgatgg aacctggaaa aaaa 1858 2 543 PRT Homo sapiens 2 Met Ser Arg GluSer Asp Val Glu Ala Gln Gln Ser His Gly Ser Ser 1 5 10 15 Ala Cys SerGln Pro His Gly Ser Val Thr Gln Ser Gln Gly Ser Ser 20 25 30 Ser Gln SerGln Gly Ile Ser Ser Ser Ser Thr Ser Thr Met Pro Asn 35 40 45 Ser Ser GlnSer Ser His Ser Ser Ser Gly Thr Leu Ser Ser Leu Glu 50 55 60 Thr Val SerThr Gln Glu Leu Tyr Ser Ile Pro Glu Asp Gln Glu Pro 65 70 75 80 Glu AspGln Glu Pro Glu Glu Pro Thr Pro Ala Pro Trp Ala Arg Leu 85 90 95 Trp AlaLeu Gln Asp Gly Phe Ala Asn Leu Glu Cys Val Asn Asp Asn 100 105 110 TyrTrp Phe Gly Arg Asp Lys Ser Cys Glu Tyr Cys Phe Asp Glu Pro 115 120 125Leu Leu Lys Arg Thr Asp Lys Tyr Arg Thr Tyr Ser Lys Lys His Phe 130 135140 Arg Ile Phe Arg Glu Val Gly Pro Lys Asn Ser Tyr Ile Ala Tyr Ile 145150 155 160 Glu Asp His Ser Gly Asn Gly Thr Phe Val Asn Thr Glu Leu ValGly 165 170 175 Lys Gly Lys Arg Arg Pro Leu Asn Asn Asn Ser Glu Ile AlaLeu Ser 180 185 190 Leu Ser Arg Asn Lys Val Phe Val Phe Phe Asp Leu ThrVal Asp Asp 195 200 205 Gln Ser Val Tyr Pro Lys Ala Leu Arg Asp Glu TyrIle Met Ser Lys 210 215 220 Thr Leu Gly Ser Gly Ala Cys Gly Glu Val LysLeu Ala Phe Glu Arg 225 230 235 240 Lys Thr Cys Lys Lys Val Ala Ile LysIle Ile Ser Lys Arg Lys Phe 245 250 255 Ala Ile Gly Ser Ala Arg Glu AlaAsp Pro Ala Leu Asn Val Glu Thr 260 265 270 Glu Ile Glu Ile Leu Lys LysLeu Asn His Pro Cys Ile Ile Lys Ile 275 280 285 Lys Asn Phe Phe Asp AlaGlu Asp Tyr Tyr Ile Val Leu Glu Leu Met 290 295 300 Glu Gly Gly Glu LeuPhe Asp Lys Val Val Gly Asn Lys Arg Leu Lys 305 310 315 320 Glu Ala ThrCys Lys Leu Tyr Phe Tyr Gln Met Leu Leu Ala Val Gln 325 330 335 Tyr LeuHis Glu Asn Gly Ile Ile His Arg Asp Leu Lys Pro Glu Asn 340 345 350 ValLeu Leu Ser Ser Gln Glu Glu Asp Cys Leu Ile Lys Ile Thr Asp 355 360 365Phe Gly His Ser Lys Ile Leu Gly Glu Thr Ser Leu Met Arg Thr Leu 370 375380 Cys Gly Thr Pro Thr Tyr Leu Ala Pro Glu Val Leu Val Ser Val Gly 385390 395 400 Thr Ala Gly Tyr Asn Arg Ala Val Asp Cys Trp Ser Leu Gly ValIle 405 410 415 Leu Phe Ile Cys Leu Ser Gly Tyr Pro Pro Phe Ser Glu HisArg Thr 420 425 430 Gln Val Ser Leu Lys Asp Gln Ile Thr Ser Gly Lys TyrAsn Phe Ile 435 440 445 Pro Glu Val Trp Ala Glu Val Ser Glu Lys Ala LeuAsp Leu Val Lys 450 455 460 Lys Leu Leu Val Val Asp Pro Lys Ala Arg PheThr Thr Glu Glu Ala 465 470 475 480 Leu Arg His Pro Trp Leu Gln Asp GluAsp Met Lys Arg Lys Phe Gln 485 490 495 Asp Leu Leu Ser Glu Glu Asn GluSer Thr Ala Leu Pro Gln Val Leu 500 505 510 Ala Gln Pro Ser Thr Ser ArgLys Arg Pro Arg Glu Gly Glu Ala Glu 515 520 525 Gly Ala Glu Thr Thr LysArg Pro Ala Val Cys Ala Ala Val Leu 530 535 540 3 25 DNA ArtificialSequence Description of Artificial SequencePCR Primer GSP3 3 ttttgctgatgatctttatg gctac 25 4 25 DNA Artificial Sequence Description ofArtificial Sequence PCR Primer GSP4 4 cacaggcacc acttccaaga gtttt 25 536 DNA Artificial Sequence Description of Artificial Sequence PCR Primer5 gggctcgaga gcagcgatgt ctcgggagtc ggatgt 36 6 35 DNA ArtificialSequence Description of Artificial Sequence PCR Primer 6 ggcggatcctcgagtcacaa cacagcagca cacac 35

What is claimed is:
 1. An isolated nucleic acid encoding for humancheckpoint kinase 1 (HCDS1), having the nucleic acid sequencerepresented from position 66 to 1694 of the nucleic acid sequence ofFIG. 1 (SEQ ID NO: 1).
 2. An isolated nucleic acid encoding for aprotein having the amino acid sequence of FIG. 2 (SEQ ID NO: 2), or anenzymatically active fragment thereof.
 3. A nucleic acid according toclaim 2 which is a DNA molecule.
 4. A nucleic acid according to claim 3wherein said DNA molecule is a cDNA.
 5. An isolated plasmidcorresponding to plasmid having Accession Number LMBP
 3708. 6. Anisolated plasmid corresponding to plasmid having Accession Number LMBP3710.
 7. An isolated plasmid corresponding to plasmid having AccessionNumber LMBP 3709.